Meglumine salt formulations of 1-(5,6-dichloro-1H-benzo[D]imidazol-2-yl)-1H-pyrazole-4-carboxylic acid

ABSTRACT

The meglumine salt of 1-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)-1H-pyrazole-4-carboxylic acid (compound (1)) and pharmaceutically acceptable formulations thereof are described. Such compounds may be used in pharmaceutical compositions and methods for the treatment of disease states, disorders, and conditions mediated by prolyl hydroxylase activity.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional patentapplication Ser. No. 61/551,395, filed on Oct. 25, 2011.

FIELD OF THE INVENTION

The present invention is directed to the meglumine salt of1-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)-1H-pyrazole-4-carboxylic acidand related methods of manufacture.

BACKGROUND

A family of highly conserved oxygen, iron, and 2-oxoglutarate-dependentprolyl hydroxylase (PHD) enzymes mediate the cells response to hypoxiavia post-translational modification of hypoxia-inducible factors (HIF)(Ivan et al., 2001, Science, 292:464-68; Jaakkola et al., 2001, Science,292:468-72). Under normoxic conditions, PHD catalyzes the hydroxylationof two conserved proline residues within HIF. As the affinity of PHD foroxygen is within the physiological range of oxygen and oxygen is anecessary co-factor for modifying hydroxylated HIF, PHD is inactivatedwhen oxygen tension is reduced. In this way, HIF is rapidly degradedunder normoxic conditions but accumulates in cells under hypoxicconditions or when PHD is inhibited.

Four isotypes of PHD have been described: PHD1, PHD2, PHD3, and PHD4(Epstein et al., 2001, Cell, 107:43-54; Kaelin, 2005, Annu Rev Biochem.,74:115-28; Schmid et al., 2004, J Cell Mol Med., 8:423-31). Thedifferent isotypes are ubiquitously expressed but are differentiallyregulated and have distinct physiological roles in the cellular responseto hypoxia. There is evidence that the various isotypes have differentselectivity for the three different HIF-α sub-types (Epstein et al.,supra). In terms of cellular localization, PHD1 is primarily nuclear,PHD2 is primarily cytoplasmic, and PHD3 appears to be both cytoplasmicand nuclear (Metzen E, et al. 2003, J Cell Sci., 116(7):1319-26). PHD2appears to be the predominant HIF-α prolyl hydroxylase under normoxicconditions (Ivan et al., 2002. Proc Natl Acad USA, 99(21):13459-64;Berra et al., 2003, EMBO J., 22:4082-90). The three isotypes have a highdegree of amino-acid homology and the active site of the enzyme ishighly conserved.

Targeted disruption of the PHD enzyme activity by small molecules haspotential utility in the treatment of disorders of oxygen sensing anddistribution. Examples include but are not limited to: anemia; sicklecell anemia; peripheral vascular disease; coronary artery disease; heartfailure; protection of tissue from ischemia in conditions such asmyocardial ischemia, myocardial infarction and stroke; preservation oforgans for transplant; treatment of tissue ischemia by regulating and/orrestoring blood flow, oxygen delivery and/or energy utilization;acceleration of wound healing particularly in diabetic and agedpatients; treatment of burns; treatment of infection; bone healing, andbone growth. In addition, targeted disruption of PHD is expected to haveutility in treating metabolic disorders such as diabetes, obesity,ulcerative colitis, inflammatory bowel disease and related disorderssuch as Crohn's disease. (Recent Patents on Inflammation & Allergy DrugDiscovery, 2009, 3:1-16).

HIF has been shown to be the primary transcriptional factor that leadsto increased erythropoietin production under conditions of hypoxia (Wanget al., 1993, supra). While treatment with recombinant humanerythropoietin has been demonstrated to be an effective method oftreating anemia, small molecule mediated PHD inhibition can be expectedto offer advantages over treatment with erythropoietin. Specifically,the function of other HIF gene products is necessary for hematopoesisand regulation of these factors increases the efficiency ofhematopoesis. Examples of HIF target gene products that are critical forhematopoesis include: transferrin (Rolfs et al., 1997, J Biol Chem.,272(32):20055-62), transferrin receptor (Lok et al., 1999, J Biol Chem.,274(34):24147-52; Tacchini et al., 1999, J Biol Chem., 274(34):24142-46)and ceruloplasmin (Mukhopadhyay et al., 2000, J Biol Chem.,275(28):21048-54). Hepcidin expression is also suppressed by HIF(Peyssonnaux et al., 2007, J Clin Invest., 117(7):1926-32) and smallmolecule inhibitors of PHD have been shown to reduce hepcidin production(Braliou et al., 2008, J Hepatol., 48:801-10). Hepcidin is a negativeregulator of the availability of the iron that is necessary forhematopoesis, so a reduction in hepcidin production is expected to bebeneficial to the treatment of anemia. PHD inhibition may also be usefulwhen used in conjunction with other treatments for anemia including ironsupplementation and/or exogenous erythropoietin. Studies of mutations inthe PHD2 gene occurring naturally in the human population providefurther evidence for the use of PHD inhibitors to treat anemia. Tworecent reports have shown that patients with dysfunctional mutations inthe PHD2 gene display increased erythrocytosis and elevated bloodhemoglobin (Percy et al., 2007, PNAS, 103(3):654-59; Al-Sheikh et al.,2008, Blood Cells Mol Dis., 40:160-65). In addition, a small moleculePHD inhibitor has been evaluated in healthy volunteers and patients withchronic kidney disease (U.S. Pat. App. No. US2006/0276477, Dec. 7,2006). Plasma erythropoietin was increased in a dose-dependent fashionand blood hemoglobin concentrations were increased in the chronic kidneydisease patients.

Overall accumulation of HIF under hypoxic conditions governs an adaptiveup-regulation of glycolysis, a reduction in oxidative phosphorylationresulting in a reduction in the production of hydrogen peroxide andsuperoxide, optimization of oxidative phosphorylation protecting cellsagainst ischemic damage. Thus, PHD inhibitors are expected to be usefulin organ and tissue transplant preservation (Bernhardt et al., 2007,Methods Enzymol., 435:221-45). While benefit may be achieved byadministering PHD inhibitors before harvesting organs for transplant,administration of an inhibitor to the organ/tissue after harvest, eitherin storage (e.g., cardioplegia solution) or post-transplant, may also beof therapeutic benefit.

PHD inhibitors are expected to be effective in preserving tissue fromregional ischemia and/or hypoxia. This includes ischemia/hypoxiaassociated with inter alia: angina, myocardial ischemia, stroke,ischemia of skeletal muscle. Recently, ischemic pre-conditioning hasbeen demonstrated to be a HIF-dependent phenomenon (Cai et al., 2008,Cardiovasc Res., 77(3):463-70). While the concept of pre-conditioning isbest known for its protective effects in the heart, it also applies toother tissues including but not limited to: liver, skeletal muscle,liver, lung, kidney, intestine and brain (Pasupathy et al., 2005, Eur JVasc Endovasc Surg., 29:106-15; Mallick et al., 2004, Dig Dis Sci.,49(9):1359-77). Experimental evidence for the tissue protective effectsof PHD inhibition and elevation of HIF have been obtained in a number ofanimal models including: germ-line knock out of PHD1 which conferredprotection of the skeletal muscle from ischemic insult (Aragones et al.,2008, Nat Genet., 40(2):170-80), silencing of PHD2 through the use ofsiRNA which protected the heart from ischemic insult (Natarajan et al.,2006, Circ Res., 98(1):133-40), inhibition of PHD by administeringcarbon monoxide which protected the myocardium from ischemic injury(Chin et al., 2007, Proc Natl Acad Sci. U.S.A., 104(12):5109-14),hypoxia in the brain which increased the tolerance to ischemia(Bernaudin et al., 2002, J Cereb Blood Flow Metab., 22(4):393-403). Inaddition, small molecule inhibitors of PHD protect the brain inexperimental stroke models (Siddiq et al., 2005, J Biol Chem.,280(50):41732-43). Moreover, HIF up-regulation has also been shown toprotect the heart of diabetic mice, where outcomes are generally worse(Natarajan et al., 2008, J Cardiovasc Pharmacol., 51(2):178-187). Thetissue protective effects may also be observed in Buerger's disease,Raynaud's disease, and acrocyanosis.

The reduced reliance on aerobic metabolism via the Kreb's cycle in themitochondria and an increased reliance on anaerobic glycolysis producedby PHD inhibition may have beneficial effects in normoxic tissues. It isimportant to note that PHD inhibition has also been shown to elevate HIFunder normoxic conditions. Thus, PHD inhibition produces a pseudohypoxiaassociated with the hypoxic response being initiated through HIF butwith tissue oxygenation remaining normal. The alteration of metabolismproduced by PHD inhibition can also be expected to provide a treatmentparadigm for diabetes, obesity and related disorders, includingco-morbidities.

Globally, the collection of gene expression changes produced by PHDinhibition reduce the amount of energy generated per unit of glucose andwill stimulate the body to burn more fat to maintain energy balance. Themechanisms for the increase in glycolysis are discussed above. Otherobservations link the hypoxic response to effects that are expected tobe beneficial for the treatment of diabetes and obesity. Hypoxia andhypoxia mimetics such as desferrioxamine have been shown to preventadipocyte differentiation (Lin et al., 2006, J Biol Chem.,281(41):30678-83; Carriere et al., 2004, J Biol Chem.,279(39):40462-69). Inhibition of PHD activity during the initial stagesof adipogenesis inhibits the formation of new adipocytes (Floyd et al.,2007, J Cell Biochem., 101:1545-57). Hypoxia, cobalt chloride anddesferrioxamine elevated HIF and inhibited PPAR gamma 2 nuclear hormonereceptor transcription (Yun et al., 2002, Dev Cell., 2:331-41). As PPARgamma 2 is an important signal for adipocyte differentiation, PHDinhibition can be expected to inhibit adipocyte differentiation. Theseeffects were shown to be mediated by the HIF-regulated gene DEC1/Stra13(Yun et al., supra).

Small molecular inhibitors of PHD have been demonstrated to havebeneficial effects in animal models of diabetes and obesity (Intl. Pat.App. Pub. No. WO2004/052284, Jun. 24, 2004; WO2004/052285, Jun. 24,2004). Among the effects demonstrated for PHD inhibitors in mousediet-induced obesity, db/db mouse and Zucker fa/fa rat models werelowering of: blood glucose concentration, fat mass in both abdominal andvisceral fat pads, hemoglobin A1c, plasma triglycerides, body weight aswell as changes in established disease bio-markers such as increases inthe levels of adrenomedullin and leptin. Leptin is a known HIF targetgene product (Grosfeld et al., 2002, J Biol Chem., 277(45):42953-57).Gene products involved in the metabolism in fat cells were demonstratedto be regulated by PHD inhibition in a HIF-dependent fashion (Intl. Pat.App. Pub. No. WO2004/052285, supra). These include apolipoprotein A-IV,acyl CoA thioesterase, carnitine acetyl transferase, and insulin-likegrowth factor binding protein (IGFBP)-1.

PHD inhibitors are expected to be therapeutically useful as stimulantsof vasculogenesis, angiogenesis, and arteriogenesis. These processesestablish or restore blood flow and oxygenation to the tissues underischemia and/or hypoxia conditions (Semenza et al., 2007, J CellBiochem., 102:840-47; Semenza, 2007, Exp Physiol., 92(6):988-91). It hasbeen shown that physical exercise increases HIF-1 and vascularendothelial growth factor in experimental animal models and in humans(Gustafsson et al. 2001, Front Biosci., 6:D75-89) and consequently thenumber of blood vessels in skeletal muscle. VEGF is a well-known HIFtarget gene product that is a key driver of angiogenesis (Liu et al.,supra). PHD inhibition offers a potential advantage over otherangiogenic therapies in that it stimulates a controlled expression ofmultiple angiogenic growth factors in a HIF-dependent fashion includingbut not limited to: placental growth factor (PLGF), angiopoietin-1(ANGPT1), angiopoietin-2 (ANGPT2), platelet-derived growth factor beta(PDGFB) (Carmeliet, 2004, J Intern Med., 255:538-61; Kelly et al., 2003,Circ Res., 93:1074-81) and stromal cell derived factor 1 (SDF-1)(Ceradini et al., 2004, Nat Med., 10(8):858-64). Expression ofangiopoietin-1 during angiogenesis produces leakage-resistant bloodvessels, in contrast to the vessels produced by administration of VEGFalone (Thurston et al., 1999, Science, 286:2511-14; Thurston et al.,2000, Nat Med., 6(4):460-3; Elson et al., 2001, Genes Dev.,15(19):2520-32). Stromal cell derived factor 1 (SDF-1) has been shown tobe critical to the process of recruiting endothelial progenitor cells tothe sites of tissue injury. SDF-1 expression increased the adhesion,migration and homing of circulating CXCR4-positive progenitor cells toischemic tissue. Furthermore inhibition of SDF-1 in ischemic tissue orblockade of CXCR4 on circulating cells prevents progenitor cellrecruitment to sites of injury (Ceradini et al., 2004, supra; Ceradiniet al., 2005, Trends Cardiovasc Med., 15(2):57-63). Importantly, therecruitment of endothelial progenitor cells to sites of injury isreduced in aged mice and this is corrected by interventions thatincrease HIF at the wound site (Chang et al., 2007, Circulation,116(24):2818-29). PHD inhibition offers the advantage not only ofincreasing the expression of a number of angiogenic factions but also aco-ordination in their expression throughout the angiogenesis processand recruitment of endothelial progenitor cells to ischemic tissue.

PHD inhibitors are useful in pro-angiogenic therapies, too.Adenovirus-mediated over-expression of HIF has been demonstrated toinduce angiogenesis in non-ischemic tissue of an adult animal (Kelly etal., 2003, Circ Res., 93(11):1074-81) providing evidence that therapiesthat elevate HIF, such as PHD inhibition, will induce angiogenesis.Placental growth factor (PLGF), also a HIF target gene, has been show toplay a critical role in angiogenesis in ischemic tissue (Carmeliet,2004, J Intern Med., 255(5):538-61; Luttun et al., 2002, Ann N Y AcadSci., 979:80-93). The potent pro-angiogenic effects of therapies thatelevate HIF have been demonstrated, via HIF over-expression, in skeletalmuscle (Pajusola et al., 2005, FASEB J., 19(10):1365-7; Vincent et al.,2000, Circulation, 102:2255-61) and in the myocardium (Shyu et al.,2002, Cardiovasc Res., 54:576-83). The recruitment of endothelialprogenitor cells to the ischemic myocardium by the HIF target gene SDF-1has also been demonstrated (Abbott et al., 2004, Circulation,110(21):3300-05). Thus, PHD inhibitors will likely be effective instimulating angiogenesis in the setting of tissue ischemia, particularlymuscle ischemia. Therapeutic angiogenesis produced by PHD inhibitorswill likely lead to restoring blood flow to tissues and thereforemeliorate such diseases as but not limited to angina pectoris,myocardial ischemia and infarction, peripheral ischemic disease,claudication, gastric and duodenal ulcers, ulcerative colitis, andinflammatory bowel disease.

PHD and HIF play a central role in tissue repair and regenerationincluding healing of wounds and ulcers. Recent studies have demonstratedthat an increased expression of all three PHDs at wound sites in agedmice with a resulting reduction in HIF accumulation (Chang et al.,supra). Thus, elevation of HIF in aged mice by administeringdesferrioxamine increased the degree of wound healing back to levelsobserved in young mice. Similarly, in a diabetic mouse model, HIFelevation was suppressed compared to non-diabetic litter mates (Mace etal., 2007, Wound Repair Regen., 15(5):636-45). Topical administration ofcobalt chloride, a hypoxia mimetic, or over-expression of a murine HIFthat lacks the oxygen-dependent degradation domain and thus provides fora constitutively active form of HIF, resulted in increased HIF at thewound site, increased expression of HIF target genes such as VEGF, Nos2,and Hmox1 and accelerated wound healing. The beneficial effect of PHDinhibition is not restricted to the skin and small molecule inhibitorsof PHD have recently been demonstrated to provide benefit in a mousemodel of colitis (Robinson et al., 2008, Gastroenterology,134(1):145-55).

In summary, PHD inhibition resulting in accumulation of HIF likely actsby at least four mechanisms to contribute to accelerated and morecomplete healing of wounds: 1) protection of tissue jeopardized byhypoxia and/or ischemia, 2) stimulation of angiogenesis to establish orrestore appropriate blood flow to the site, 3) recruitment ofendothelial progenitor cells to wound sites, 4) stimulation of therelease of growth factors that specifically stimulate healing andregeneration.

As PDGF is a HIF gene target (Schultz et al., 2006, Am J Physiol HeartCirc Physiol., 290(6):H2528-34; Yoshida et al., 2006, J Neurooncol.,76(1):13-21), PHD inhibition likely increases the expression ofendogenous PDGF and produces a similar or more beneficial effect tothose produced with PDGF alone. Studies in animals have shown thattopical application of PDGF results in increased wound DNA, protein, andhydroxyproline amounts; formation of thicker granulation and epidermaltissue; and increased cellular repopulation of wound sites. PDGF exertsa local effect on enhancing the formation of new connective tissue. Theeffectiveness of PHD inhibition is likely greater than that produced byPDGF due to the additional tissue protective and pro-angiogenic effectsmediated by HIF.

The beneficial effects of inhibition of PHD extends not only toaccelerated wound healing in the skin and colon but also to the healingof other tissue damage including but not limited to gastrointestinalulcers, skin graft replacements, burns, chronic wounds and frost bite.

Stem cells and progenitor cells are found in hypoxic niches within thebody and hypoxia regulates their differentiation and cell fate (Simon etal., 2008, Nat Rev Mol Cell Biol., 9:285-96). Thus, PHD inhibitors maybe useful to maintain stem cells and progenitor cells in a pluripotentstate and to drive differentiation to desired cell types. Stem cells maybe useful in culturing and expanding stem cell populations and may holdcells in a pluripotent state while hormones and other factors areadministered to the cells to influence the differentiation and cellfate.

A further use of PHD inhibitors in the area of stem cell and progenitorcell therapeutics relates to the use of PHD inhibitors to conditionthese cells to withstand the process of implantation into the body andto generate an appropriate response to the body to make the stem celland progenitor cell implantation viable (Hu et al., 2008, J ThoracCardiovasc Surg., 135(4):799-808). More specifically PHD inhibitors mayfacilitate the integration of stem cells and draw in an appropriateblood supply to sustain the stem cells once they are integrated. Thisblood vessel formation will also function to carry hormones and otherfactors released from these cells to the rest of the body.

PHD inhibitors may also be useful in the treatment of infection(Peyssonnaux et al., 2005, J Invest Dermatol., 115(7):1806-15;Peyssonnaux et al., 2008 J Invest Dermatol., 2008 August;128(8):1964-8). HIF elevation has been demonstrated to increase theinnate immune response to infection in phagocytes and in keratinocytes.Phagocytes in which HIF is elevated show increased bacteriacidalactivity, increased nitric oxide production and increased expressed ofthe anti-bacterial peptide cathelicidin. These effects may also beuseful in treating infection from burns.

HIF has also been shown to be involved in bone growth and healing(Pfander D et al., 2003 J Cell Sci., 116(Pt 9):1819-26., Wang et al.,2007 J Clin Invest., 17(6):1616-26.) and may therefore be used to healor prevent fractures. HIF stimulates of glycolysis to provide energy toallow the synthesis of extracellular matrix of the epiphysealchondrocytes under a hypoxic environment. HIF also plays a role indriving the release of VEGF and angiogenesis in bone healing process.The growth of blood vessels into growing or healing bone can be the ratelimiting step in the process.

Small molecules inhibitors of PHD have been described in the literature,which include, but are not limited to, imidazo[1,2-a]pyridinederivatives (Warshakoon et al., 2006, Bioorg Med Chem Lett.,16(21):5598-601), substituted pyridine derivatives (Warshakoon et al.,2006, Bioorg Med Chem Lett., 16(21):5616-20), pyrazolopyridines(Warshakoon et al., 2006, Bioorg Med Chem Lett., 16(21):5687-90),bicyclic heteroaromatic N-substituted glycine derivatives (Intl. Pat.App. Pub. No. WO2007/103905, Sep. 13, 2007), quinoline based compounds(Intl. Pat. App. Pub. No. WO2007/070359, Jun. 21, 2007),pyrimidinetrione N-substituted glycine derivatives (Intl. Pat. App. Pub.No. WO2007/150011, Dec. 27, 2007), substituted aryl or heteroaryl amidecompounds (U.S. Pat. App. Pub. No. US 2007/0299086, Dec. 27, 2007) andsubstituted 4-hydroxypyrimidine-5-carboxamides (Intl. Pat. App. Pub. No.WO2009/117269, Sep. 24, 2009).

SUMMARY OF THE INVENTION

The invention is directed to the general and preferred embodimentsdefined, as set forth herein. Preferred and exemplary features of theinvention will be apparent from the detailed description below and withreference to the drawing figures.

In its many embodiments, the present invention relates to a novel saltof an inhibitor of prolyl hydroxylase (PHD) enzymes, and a method oftreatment, prevention, inhibition or amelioration of one or morediseases disorders associated with PHD enzymes is provided.

More particularly, the present invention relates to the meglumine saltof a compound of the following formula:

and related methods of preparation or manufacture of the compound.

In another embodiment, the present invention relates to the hydratedform of the meglumine salt of compound (1).

Additional embodiments and advantages of the invention will becomeapparent from the detailed discussion, schemes, examples, and claimsbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the pH-dependency of saturation of compound (1) insolution.

FIG. 2 shows PXRD data of the meglumine salt of compound (1).

FIG. 3 shows DSC, TGA and x-ray data of the meglumine salt of compound

FIG. 4 shows: (A) the single crystal structure of the meglumine salt ofcompound (1); and (B) the experimental and stimulated powder pattern ofa single crystal for the meglumine salt of compound (1).

FIG. 5 shows the percentage stimulation of HIF1-α upon exposure offormulations of the meglumine salt of compound (1).

FIG. 6 shows plasma levels (systemic burden) in wounded mice aftertopical application of formulations of the meglumine salt of compound(1).

FIG. 7 shows the correlation of the flux of compound (1) across skin(human dermis) and an artificial membrane using a Franz diffusion cellupon application of a formulation of meglumine salt of compound (1).

FIG. 8 shows no to very low irritation caused by application of aformulation of the meglumine salt of compound (1) as tested in a HET-CAMassay.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a novel salt of a compound of the followingformula:

that is a inhibitor of prolyl hydroxylase (PHD) enzymes, andcompositions thereof for the treatment, amelioration or inhibition ofdisorders and diseases related to the modulation of a prolyl hydroxylaseenzyme. The present invention also relates to methods of making such acompound, pharmaceutical compositions, pharmaceutically acceptablesalts, pharmaceutically acceptable prodrugs, and pharmaceutically activemetabolites thereof.

A) Terms

The present invention is best understood by reference to the followingdefinitions, the drawings and exemplary disclosure provided herein.

The terms “comprising”, “containing”, and “including,” are used hereinin their open, non-limiting sense.

“Administering” or “administration” means providing a drug to a patientin a manner that is pharmacologically useful.

“Composition” means a product containing a compound of the presentinvention (such as a product comprising the specified ingredients in thespecified amounts, as well as any product which results, directly orindirectly, from such combinations of the specified ingredients in thespecified amounts).

“Compound” or “drug” means a compound of Formula (1) or pharmaceuticallyacceptable forms thereof.

“Forms” means various isomers and mixtures of one or more compounds ofFormula (1) and salts or hydrates thereof. The term “isomer” refers tocompounds that have the same composition and molecular weight but differin physical and/or chemical properties. Such substances have the samenumber and kind of atoms but differ in structure. The structuraldifference may be in constitution (geometric isomers) or in an abilityto rotate the plane of polarized light (stereoisomers). The term“stereoisomer” refers to isomers of identical constitution that differin the arrangement of their atoms in space. Enantiomers anddiastereomers are stereoisomers wherein an asymmetrically substitutedcarbon atom acts as a chiral center. The term “chiral” refers to amolecule that is not superposable on its mirror image, implying theabsence of an axis and a plane or center of symmetry.

The term “hypoxia” or “hypoxic disorder” refers to a condition wherethere is an insufficient level of oxygen provided in the blood or totissues and organs. Hypoxic disorders can occur through a variety ofmechanisms including where there is an insufficient capacity of theblood to carry oxygen (i.e. anemia), where there is an inadequate flowof blood to the tissue and/or organ caused by either heart failure orblockage of blood vessels and/or arteries (i.e. ischemia), where thereis reduced barometric pressure (i.e. elevation sickness at highaltitudes), or where dysfunctional cells are unable to properly make useof oxygen (i.e. hystotoxic conditions). Accordingly, one of skill in theart would readily appreciate the present invention to be useful in thetreatment of a variety of hypoxic conditions including anemia, heartfailure, coronary artery disease, thromboembolism, stroke, angina andthe like.

“Patient” or “subject” means an animal, preferably a mammal, morepreferably a human, in need of therapeutic intervention.

“Pharmaceutically acceptable” means molecular entities and compositionsthat are of sufficient purity and quality for use in the formulation ofa composition or medicament of the present invention. Since both humanuse (clinical and over-the-counter) and veterinary use are equallyincluded within the scope of the present invention, a formulation wouldinclude a composition or medicament for either human or veterinary use.

“Pharmaceutically acceptable excipient” refers to a substance that isnon-toxic, biologically tolerable, and otherwise biologically suitablefor administration to a subject, such as an inert substance, added to apharmacological composition or otherwise used as a vehicle, carrier, ordiluent to facilitate administration of an agent and that is compatibletherewith. Examples of excipients include calcium carbonate, calciumphosphate, various sugars and types of starch, cellulose derivatives,gelatin, vegetable oils, and polyethylene glycols.

“Pharmaceutically acceptable salt” means an acid or base salt of thecompounds of the invention that is of sufficient purity and quality foruse in the formulation of a composition or medicament of the presentinvention and are tolerated and sufficiently non-toxic to be used in apharmaceutical preparation. Suitable pharmaceutically acceptable saltsinclude acid addition salts which may, for example, be formed byreacting the drug compound with a suitable pharmaceutically acceptableacid such as hydrochloric acid, sulfuric acid, fumaric acid, maleicacid, succinic acid, acetic acid, benzoic acid, citric acid, tartaricacid, carbonic acid or phosphoric acid.

The term “solvates” means those compounds that are formed from theinteraction or complexation of such compounds with one or more solventmolecule, either in solution or in solid or crystalline form. The term“hydrates” mean solvates, wherein the solvent is water.

“Therapeutically effective amount” means that amount of compound thatelicits the biological or medicinal response in a tissue system, animalor human, that is being sought by a researcher, veterinarian, medicaldoctor, or other clinician, which includes therapeutic alleviation ofthe symptoms of the disease or disorder being treated.

The term “treating” as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, lessening theseverity of, or preventing the disorder or condition to which such termapplies, or one or more symptoms of such disorder or condition. The term“treatment”, as used herein, unless otherwise indicated, refers to theact of treating.

B) Compounds

The present invention relates to novel salts of compound of Formula (1).In particular, the invention relates to the meglumine salt of compoundof Formula (1). In general, the invention relates to all compounds thatupon administration to patients in need of treatment of disorders anddiseases related to the modulation of a prolyl hydroxylase enzyme.

Some embodiments of the invention include hydrates, solvates orpolymorphs of such compounds, and mixtures thereof, even if such formsare not explicitly stated in the present specification. Preferably, someembodiments of compounds of Formula (1) or pharmaceutically acceptablesalts thereof include solvates. More preferably, some embodiments ofcompounds of Formula (1) or pharmaceutically acceptable salts thereofinclude hydrates.

Yet another embodiment of the invention includes crystalline forms ofcompounds of Formula (1) or pharmaceutically acceptable salts ofcompounds of Formula (1) may be obtained as co-crystals.

In certain embodiments of the invention, compounds of Formula (1) wereobtained in a crystalline form. In other embodiments, crystalline formsof compounds of Formula (1) were cubic in nature. In other embodiments,pharmaceutically acceptable salts of compounds of Formula (1) wereobtained in a crystalline form. In still other embodiments, compounds ofFormula (1) were obtained in one of several polymorphic forms, as amixture of crystalline forms, as a polymorphic form, or as an amorphousform. In other embodiments, compounds of Formula (1) convert in solutionbetween one or more crystalline forms and/or polymorphic forms.

Drug compounds of the present invention also include a mixture ofstereoisomers, or each pure or substantially pure isomer. For example,the present compound may optionally have one or more asymmetric centersat a carbon atom containing any one substituent. Therefore, the compoundmay exist in the form of enantiomer or diastereomer, or a mixturethereof. When the present compound contains a double bond, the presentcompound may exist in the form of geometric isomerism (cis-compound,trans-compound), and when the present compound contains an unsaturatedbond such as carbonyl, then the present compound may exist in the formof a tautomer, and the present compound also includes these isomers or amixture thereof. The starting compound in the form of a racemic mixture,enantiomer or diastereomer may be used in the processes for preparingthe present compound. When the present compound is obtained in the formof a diastereomer or enantiomer, they can be separated by a conventionalmethod such as chromatography or fractional crystallization. Inaddition, the present compound includes an intramolecular salt, hydrate,solvate or polymorphism thereof. Suitable drug compounds are those thatexert a local physiological effect, or a systemic effect, either afterpenetrating the mucosa, dermis or—in the case of oraladministration—after transport to the gastrointestinal tract withsaliva.

The invention further relates to pharmaceutically acceptable salts ofcompounds of Formula (1) and methods of using such salts. Apharmaceutically acceptable salt refers to a salt of a free acid or baseof the compound that is non-toxic, biologically tolerable, or otherwisebiologically suitable for administration to the subject. See, generally,S. M. Berge, et al., “Pharmaceutical Salts”, J. Pharm. Sci., 1977,66:1-19, and Handbook of Pharmaceutical Salts, Properties, Selection,and Use, 2002, Stahl and Wermuth, Eds., Wiley-VCH and VHCA, Zurich.Preferred pharmaceutically acceptable salts are those that arepharmacologically effective and suitable for contact with the tissues ofpatients without undue toxicity, irritation, or allergic response. Acompound may possess a sufficiently acidic group, a sufficiently basicgroup, or both types of functional groups, and accordingly react with anumber of inorganic or organic bases, and inorganic and organic acids,to form a pharmaceutically acceptable salt. Examples of pharmaceuticallyacceptable salts include sulfates, pyrosulfates, bisulfates, sulfites,bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates,propionates, decanoates, caprylates, acrylates, formates, isobutyrates,caproates, heptanoates, propiolates, oxalates, malonates, succinates,suberates, sebacates, fumarates, maleates, butyne-1,4-dioates,hexyne-1,6-dioates, benzoates, chlorobenzoates, methyl benzoates,dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates,sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, methane-sulfonates, propanesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates.

In the presence of a basic nitrogen, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, hydriodic acid, perchloricacid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoricacid, and the like, or with an organic acid, such as acetic acid,trifluoroacetic acid, phenylacetic acid, propionic acid, stearic acid,lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, malic acid,pamoic acid, isethionic acid, succinic acid, valeric acid, fumaric acid,saccharinic acid, malonic acid, pyruvic acid, oxalic acid, glycolicacid, salicylic acid, oleic acid, palmitic acid, lauric acid, apyranosidyl acid, such as glucuronic acid or galacturonic acid, analpha-hydroxy acid, such as mandelic acid, citric acid, or tartaricacid, an amino acid, such as aspartic acid or glutamic acid, an aromaticacid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid, orcinnamic acid, a sulfonic acid, such as laurylsulfonic acid,benzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonicacid, methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic,a cyclohexanesulfamic acid, any compatible mixture of acids such asthose given as examples herein, and any other acid and mixture thereofthat are regarded as equivalents or acceptable substitutes in light ofthe ordinary level of skill in this technology.

In the presence of an acid group, such as a carboxylic acid or sulfonicacid, the desired pharmaceutically acceptable salt may be prepared byany suitable method, for example, treatment of the free acid with aninorganic or organic base, such as an amine (primary, secondary ortertiary), an alkali metal hydroxide, alkaline earth metal hydroxide,any compatible mixture of bases such as those given as examples herein,and any other base and mixture thereof that are regarded as equivalentsor acceptable substitutes in light of the ordinary level of skill inthis technology. Illustrative examples of suitable salts include organicsalts derived from amino acids, such as glycine and arginine, ammonia,carbonates, bicarbonates, primary, secondary, and tertiary amines, andcyclic amines, such as benzylamines, pyrrolidines, piperidine,morpholine, and piperazine, and inorganic salts derived from sodium,calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum,and lithium. Representative organic or inorganic bases further includebenzathine, chloroprocaine, choline, diethanolamine, ethylenediamine,meglumine, and procaine.

The invention also relates to pharmaceutically acceptable prodrugs ofthe compounds, and treatment methods employing such pharmaceuticallyacceptable prodrugs. The term “prodrug” means a precursor of adesignated compound that, following administration to a subject yieldsthe compound in vivo via a chemical or physiological process such assolvolysis or enzymatic cleavage, or under physiological conditions. A“pharmaceutically acceptable prodrug” is a prodrug that is non-toxic,biologically tolerable, and otherwise biologically suitable foradministration to the subject. Illustrative procedures for the selectionand preparation of suitable prodrug derivatives are described, forexample, in “Design of Prodrugs”, ed. H. Bundgaard, 1985, Elsevier.

Additional types of prodrugs may be produced, for instance, byderivatizing free carboxyl groups of structures of the compound asamides or alkyl esters. Examples of amides include those derived fromammonia, primary alkyl amines and secondary di-alkyl amines. Secondaryamines include 5- or 6-membered heterocycloalkyl or heteroaryl ringmoieties. Examples of amides include those that are derived fromammonia, alkyl primary amines, and di-alkyl amines. Examples of estersof the invention include alkyl, cycloalkyl, phenyl, and phenyl-alkylesters. Preferred esters include methyl esters. Prodrugs may also beprepared by derivatizing free hydroxy groups using groups includinghemisuccinates, phosphate esters, dimethylaminoacetates, andphosphoryloxymethyloxycarbonyls, following procedures such as thoseoutlined in Fleisher et al., Adv. Drug Delivery Rev., 1996, 19:115-130.Carbamate derivatives of hydroxy and amino groups may also yieldprodrugs. Carbonate derivatives, sulfonate esters, and sulfate esters ofhydroxy groups may also provide prodrugs. Derivatization of hydroxygroups as acyloxy-methyl and acyloxy-ethyl ethers, wherein the acylgroup may be an alkyl ester, optionally substituted with one or moreether, amine, or carboxylic acid functionalities, or where the acylgroup is an amino acid ester as described above, is also useful to yieldprodrugs. Prodrugs of this type may be prepared as described inGreenwald, et al., J. Med. Chem., 1996, 39 (10):1938-40. Free amines canalso be derivatized as amides, sulfonamides or phosphonamides. All ofthese prodrug moieties may incorporate groups including ether, amine,and carboxylic acid functionalities.

The present invention also relates to pharmaceutically activemetabolites of the compounds of Formula (1), which may also be used inthe methods of the invention. A “pharmaceutically active metabolite”means a pharmacologically active product of metabolism in the body ofthe compound or salt thereof. Prodrugs and active metabolites of acompound may be determined using routine techniques known or availablein the art. See, e.g., Bertolini, et al., J. Med. Chem., 1997,40:2011-2016; Shan, et al., J. Pharm. Sci., 1997, 86 (7):765-767;Bagshawe, Drug Dev. Res., 1995, 34:220-230; Bodor, Adv. Drug Res., 1984,13:224-331; Bundgaard, Design of Prodrugs, 1985, Elsevier Press; andLarsen, Design and Application of Prodrugs, Drug Design and Development,1991, Krogsgaard-Larsen, et al., eds., Harwood Academic Publishers.

C) Pharmaceutical Compositions

In particular embodiments of the invention, the salts of compounds ofFormula (1), more particularly the meglumine salt, are used alone, or incombination with one or more additional ingredients, to formulatepharmaceutical compositions. A pharma-ceutical composition comprises aneffective amount of at least one compound in accordance with theinvention.

The disclosure also provides compositions (including pharmaceuticalcompositions) comprising a compound or derivatives described herein, andone or more of pharmaceutically acceptable carrier, excipient, anddiluent. In certain embodiments of the invention, a composition may alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. In a specific embodiment, the pharmaceutical composition ispharmaceutically acceptable for administration to a human. In certainembodiments, the pharmaceutical composition comprises a therapeuticallyor prophylactically effective amount of a compound or derivativedescribed herein. The amount of a compound or derivative of theinvention that will be therapeutically or prophylactically effective canbe determined by standard clinical techniques. Exemplary effectiveamounts are described in more detail in below sections. In certainembodiments of the invention, a composition may also contain astabilizer. A stabilizer is a compound that reduces the rate of chemicaldegradation of the composition of compound (1). Suitable stabilizersinclude, but are not limited to, antioxidants, such as ascorbic acid, pHbuffers, or salt buffers.

The pharmaceutical compositions can be in any form suitable foradministration to a subject, preferably a human subject. In certainembodiments, the compositions are in the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, and sustained-releaseformulations. The compositions may also be in particular unit dosageforms. Examples of unit dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; ointments;cataplasms (poultices); pastes; powders; dressings; creams; plasters;solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels;liquid dosage forms suitable for oral or mucosal administration to apatient, including suspensions (e.g., aqueous or non aqueous liquidsuspensions, oil in water emulsions, or a water in oil liquidemulsions), solutions, and elixirs; liquid dosage forms suitable forparenteral administration to a subject; and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for parenteral administration to a subject.

In a specific embodiment, the subject is a mammal such as a cow, horse,sheep, pig, fowl, cat, dog, mouse, rat, rabbit, or guinea pig. In apreferred embodiment, the subject is a human. Preferably, thepharmaceutical composition is suitable for veterinary and/or humanadministration. In accordance with this embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly for use in humans.

Suitable pharmaceutical carriers for use in the compositions are sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin. In a specific embodiment, the oil ispeanut oil, soybean oil, mineral oil, or sesame oil. Water is apreferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Further examples of suitable pharmaceuticalcarriers are known in the art, e.g., as described in Remington'sPharmaceutical Sciences (1990) 18th ed. (Mack Publishing, Easton Pa.).

Suitable excipients for use in the compositions include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, and ethanol. Whether a particularexcipient is suitable for incorporation into a pharmaceuticalcomposition depends on a variety of factors well known in the artincluding, but not limited to, the route of administration and thespecific active ingredients in the composition.

Pharmaceutical compositions comprising the compounds or derivativesdescribed herein, or their pharmaceutically acceptable salts andsolvates, are formulated to be compatible with the intended route ofadministration. The formulations are preferably for topicaladministration, but can be for administration by other means such as byinhalation or insufflation (either through the mouth or the nose),intradermal, oral, subcutaneous, buccal, parenteral, vaginal, or rectal.Preferably, the compositions are also formulated to provide increasedchemical stability of the compound during storage and transportation.The formulations may be lyophilized or liquid formulations.

D) Administration

A compound or derivative described herein, or a pharmaceuticallyacceptable salt thereof, is preferably administered as a component of acomposition that optionally comprises a pharmaceutically acceptablevehicle. The compound or derivative is preferably administered orally.Another preferred method of administration is via topical application ofthe compound or derivative.

In certain embodiments, the compound or derivative is administered byany other convenient route, for example, by absorption through skin,epithelial or mucocutaneous linings (e.g., (epi-)dermis, oral mucosa,rectal, and intestinal mucosa). Methods of administration include butare not limited to parenteral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intranasal, intracerebral, intravaginal, transdermal,rectally, by inhalation, or topically, particularly to the ears, nose,eyes, or skin. In most instances, administration will result in therelease of the compound or derivative into the bloodstream. In preferredembodiments, the compound or derivative is delivered orally.

Furthermore, the invention relates to methods of using the compoundsdescribed herein to treat subjects diagnosed with or suffering from adisease, disorder, or condition mediated by prolyl hydroxylase, such as:anemia, vascular disorders, metabolic disorders, and wound healing.

In a preferred embodiment, compounds of the present invention are usefulin the treatment or prevention of anemia comprising treatment of anemicconditions associated with chronic kidney disease, polycystic kidneydisease, aplastic anemia, autoimmune hemolytic anemia, bone marrowtransplantation anemia, Churg-Strauss syndrome, Diamond Blackfan anemia,Fanconi's anemia, Felty syndrome, graft versus host disease,hematopoietic stem cell transplantation, hemolytic uremic syndrome,myelodysplastic syndrome, nocturnal paroxysmal hemoglobinuria,osteomyelofibrosis, pancytopenia, pure red-cell aplasia, purpuraSchoenlein-Henoch, refractory anemia with excess of blasts, rheumatoidarthritis, Shwachman syndrome, sickle cell disease, thalassemia major,thalassemia minor, thrombocytopenic purpura, anemic or non-anemicpatients undergoing surgery, anemia associated with or secondary totrauma, sideroblastic anemia, anemic secondary to other treatmentincluding: reverse transcriptase inhibitors to treat HIV, corticosteroidhormones, cyclic cisplatin or non-cisplatin-containingchemotherapeutics, vinca alkaloids, mitotic inhibitors, topoisomerase IIinhibitors, anthracyclines, alkylating agents, particularly anemiasecondary to inflammatory, aging and/or chronic diseases. PHD inhibitionmay also be used to treat symptoms of anemia including chronic fatigue,pallor and dizziness.

In another preferred embodiment, molecules of the present invention areuseful for the treatment or prevention of diseases of metabolicdisorders, including but not limited to diabetes and obesity. In anotherpreferred embodiment, molecules of the present invention are useful forthe treatment or prevention of vascular disorders. These include but arenot limited to hypoxic or wound healing related diseases requiringpro-angiogenic mediators for vasculogenesis, angiogenesis, andarteriogenesis

In treatment methods according to the invention, an effective amount ofa pharmaceutical agent according to the invention is administered to asubject suffering from or diagnosed as having such a disease, disorder,or condition. An “effective amount” means an amount or dose sufficientto generally bring about the desired therapeutic or prophylactic benefitin patients in need of such treatment for the designated disease,disorder, or condition. Effective amounts or doses of the compounds ofthe present invention may be ascertained by routine methods such asmodeling, dose escalation studies or clinical trials, and by taking intoconsideration routine factors, e.g., the mode or route of administrationor drug delivery, the pharmacokinetics of the compound, the severity andcourse of the disease, disorder, or condition, the subject's previous orongoing therapy, the subject's health status and response to drugs, andthe judgment of the treating physician. An example of a dose is in therange of from about 0.001 to about 200 mg of compound per kg ofsubject's body weight per day, preferably about 0.05 to 100 mg/kg/day,or about 1 to 35 mg/kg/day, in single or divided dosage units (e.g.,BID, TID, QID). For a 70-kg human, an illustrative range for a suitabledosage amount is from about 0.05 to about 7 g/day, or about 0.2 to about2.5 g/day.

Oral tablets may include a compound according to the invention mixedwith pharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservative agents.Suitable inert fillers include sodium and calcium carbonate, sodium andcalcium phosphate, lactose, starch, sugar, glucose, methyl cellulose,magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquidoral excipients include ethanol, glycerol, water, and the like. Starch,polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystallinecellulose, and alginic acid are suitable disintegrating agents. Bindingagents may include starch and gelatin. The lubricating agent, ifpresent, may be magnesium stearate, stearic acid or talc. If desired,the tablets may be coated with a material such as glyceryl monostearateor glyceryl distearate to delay absorption in the gastrointestinaltract, or may be coated with an enteric coating.

Capsules for oral administration include hard and soft gelatin capsules.To prepare hard gelatin capsules, compounds of the invention may bemixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsulesmay be prepared by mixing the compound of the invention with water, anoil such as peanut oil or olive oil, liquid paraffin, a mixture of monoand di-glycerides of short chain fatty acids, polyethylene glycol 400,or propylene glycol.

Liquids for oral administration may be in the form of suspensions,solutions, emulsions or syrups or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid compositions may optionally contain: pharmaceutically-acceptableexcipients such as suspending agents (for example, sorbitol, methylcellulose, sodium alginate, gelatin, hydroxyethylcellulose,carboxymethylcellulose, aluminum stearate gel and the like); non-aqueousvehicles, e.g., oil (for example, almond oil or fractionated coconutoil), propylene glycol, ethyl alcohol, or water; preservatives (forexample, methyl or propyl p-hydroxybenzoate or sorbic acid); wettingagents such as lecithin; and, if desired, flavoring or coloring agents.

The active agents of this invention may also be administered by non-oralroutes. For example, the compositions may be formulated for rectaladministration as a suppository. For parenteral use, includingintravenous, intramuscular, intraperitoneal, or subcutaneous routes, thecompounds of the invention may be provided in sterile aqueous solutionsor suspensions, buffered to an appropriate pH and isotonicity or inparenterally acceptable oil. Suitable aqueous vehicles include Ringer'ssolution and isotonic sodium chloride. Such forms will be presented inunit-dose form such as ampules or disposable injection devices, inmulti-dose forms such as vials from which the appropriate dose may bewithdrawn, or in a solid form or pre-concentrate that can be used toprepare an injectable formulation. Illustrative infusion doses may rangefrom about 1 to 1000 μg/kg/minute of compound, admixed with apharmaceutical carrier over a period ranging from several minutes toseveral days.

For topical administration, the compounds may be mixed with apharmaceutical carrier at a concentration of about 0.1% to about 10% ofdrug to vehicle. Examples include lotions, creams, ointments and thelike and can be formulated by known methods. Another mode ofadministering the compounds of the invention may utilize a patchformulation to affect transdermal delivery.

A salt-selection evaluation was carried to identify a salt of compound(1) with properties most suitable for development. The criteriaconsidered essential for the selection process were crystallinity, formreproducibility from a recrystallization process, chemical and physicalstability under accelerated conditions and adequate solubility tosupport both drug substance and drug product development.

In embodiments, the compound is formulated into dosage forms suitablefor administration to patients in need thereof. The processes andequipment for preparing drug and carrier particles are disclosed inPharmaceutical Sciences, Remington, 1985, 17th Ed., 1585-1594; ChemicalEngineers Handbook, Perry, 1984, 6th Ed., pp. 21-13 to 21-19 (1984);Parrot et al., 1974, J. Pharm. Sci., 61(6): 813-829; and Hixon et al.,1990, Chem. Engineering, pp. 94-103.

The amount of compound incorporated in the dosage forms of the presentinvention may generally vary from about 10% to about 90% by weight ofthe composition depending upon the therapeutic indication and thedesired administration period, e.g., every 12 hours, every 24 hours, andthe like. Depending on the dose of compound desired to be administered,one or more of the dosage forms can be administered. Depending upon theformulation, the compound will preferably be in the form of an acetatesalt or free base form.

Further, this invention also relates to a pharmaceutical composition ora pharmaceutical dosage form as described hereinbefore for use in amethod of therapy or diagnosis of the human or non-human animal body.

This invention also relates to a pharmaceutical composition for use inthe manufacture of a pharmaceutical dosage form for oral administrationto a mammal in need of treatment, characterized in that said dosage formcan be administered at any time of the day independently of the foodtaken in by said mammal.

This invention also relates to a method of therapy or diagnosis of thehuman or non-human animal body that comprises administering to said bodya therapeutically or diagnostically effective dose of a pharmaceuticalcomposition described herein.

This invention also relates to a pharmaceutical package suitable forcommercial sale comprising a container, a dosage form as describedherein, and associated with said package written matter non-limited asto whether the dosage form can be administered with or without food.

The following formulation examples are illustrative only and are notintended to limit the scope of the inventions in any way.

EXAMPLES

Five versions of compound (1), namely the free acid, sodium, potassium,tromethamine and meglumine salts were produced and their physicalproperties and manufacturability potential guided the selection of anpreferred form of the compound.

E) Example Synthesis

To obtain the compounds described in the examples below and theircorresponding analytical data, the following experimental and analyticalprotocols were adhered to unless otherwise indicated. Unless otherwisestated, reaction mixtures were magnetically stirred at room temperature(rt), solutions were generally “dried” over a drying agent such asNa₂SO₄ or MgSO₄, and mixtures, solutions, and extracts were typically“concentrated” on a rotary evaporator under reduced pressure.

Data Analysis Setup

Thin-layer chromatography (TLC) was performed using Merck silica gel 60F₂₅₄ 2.5 cm×7.5 cm 250 μm or 5.0 cm×10.0 cm 250 μm pre-coated silica gelplates. Preparative thin-layer chromatography was performed using EMScience silica gel 60 F₂₅₄ 20 cm×20 cm 0.5 mm pre-coated plates with a20 cm×4 cm concentrating zone.

Normal-phase flash column chromatography (FCC) was performed on silicagel (SiO₂) eluting with hexanes/ethyl acetate, unless otherwise noted,whereas reversed-phase HPLC was performed on a Hewlett Packard HPLCSeries 1100, with a Phenomenex Luna C₁₈ (5 μm, 4.6×150 mm) column, anddetection was done at λ=230, 254 and 280 nm with a gradient of 10 to 99%acetonitrile/water (0.05% trifluoroacetic acid) over 5.0 min with a flowrate of 1 mL/min. Alternately, preparative HPLC purification wasperformed on a Gilson automated HPLC system running Gilson Unipoint LCsoftware with UV peak detection done at λ=220 nm and fitted with areverse phase YMC-Pack ODS-A (5 μm, 30×250 mm) column; mobile gradientof 10-99% of acetonitrile/water (0.05% trifluoroacetic acid) over 15-20min and flow rates of 10-20 mL/min.

Mass spectra (MS) were obtained on an Agilent series 1100 MSD equippedwith a ESI/APCI positive and negative multimode source unless otherwiseindicated, and nuclear magnetic resonance (NMR) spectra were obtained onBruker model DRX spectrometers with the ¹H NMR data showing chemicalshifts in ppm downfield of the tetramethylsilane reference (apparentmultiplicity, coupling constant J in Hz, integration).

Example 1 Free acid of1-(5,6-Dichloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid(compound (1))

Method A:

The free acid of compound (1) was prepared by using2,5,6-trichloro-1H-benzoimidazole and 1H-pyrazole-4-carboxylic acid. MS(ESI/CI): mass calculated for C₁₁H₆Cl₂N₄O₂, 297.1; m/z found, 296.0[M−H]⁻. ¹H NMR (500 MHz, DMSO-d₆): 14.18-12.52 (br s, 2H), 8.89 (d,J=0.5 Hz, 1H), 8.31 (d, J=0.5 Hz, 1H), 7.80 (s, 2H).

Method B:

Step A: 5,6-Dichloro-1,3-dihydro-benzoinnidazol-2-one: To the solutionof 4,5-dichloro-benzene-1,2-diamine (25 g, 0.14 mol) in dry DMF (200mL), was added CDI (23 g, 0.14 mol) as the solid. The reaction solutionwas stirred at room temperature for 1 hour, then water (500 mL) wasadded. The precipitated solid was collected by filtration, washed withwater, dried thoroughly to afford the titled compound (26.0 g, 90%). Thecrude product was used in the following reaction without furtherpurification.

Step B: 2,5,6-Trichloro-1H-benzoimidazole: Thoroughly dried5,6-dichloro-1,3-dihydro-benzoimidazol-2-one (28.4 g, 0.14 mol) wassuspended in POCl₃ (75 mL). The reaction solution was heated to refluxtemperature for 3 hours and cooled to room temperature. The solution waspoured into crushed ice/water (1.5 L) slowly with sufficient stirring.The solution was neutralized to pH=7.0 with NaOH. The precipitated solidwas collected by filtration, washed with water, and dried to afford thetitle compound (27.9 g, 90%). The crude product was used in thefollowing reaction without further purification.

Step C:1-(5,6-Dichloro-1-dimethylsulfamoyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid ethyl ester. 2,5,6-Trichloro-1H-benzoimidazole 2 (27.6 g, 0.125mol) was dissolved in dry DMF (200 mL) and then K₂CO₃ (20.7 g, 0.15 mol)and dimethylsulfamoyl chloride (17.9 g, 0.125 mol) were added. Thereaction mixture was stirred at room temperature for 16 hours. HPLCanalysis showed the complete formation of2,5,6-trichloro-benzoimidazole-1-sulfonic acid dimethylamide. In thesame pot, without isolation of 2,5,6-trichloro-benzoimidazole-1-sulfonicacid dimethylamide, was added 1H-pyrazole-4-carboxylic acid ethyl ester(17.5 g, 0.125 mol) and K₂CO₃ (20.7 g, 0.15 mol). The reaction mixturewas stirred at 70° C. for 4 hours and water (500 mL) was added while thereaction solution was still hot. The reaction solution was cooled toroom temperature. The precipitated solid was collected via filtration,washed with water and dried. The crude product was used in the followingreaction without further purification.

Step D: 1-(5,6-Dichloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid. Crude1-(5,6-Dichloro-1-dimethylsulfamoyl-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylicacid ethyl ester was dissolved in THF (125 mL) and LiOH—H₂O (21 g, 0.5mol) in water (250 mL) was added. The reaction mixture was stirred atreflux temperature for 2 hours and cooled to room temperatue.Concentrated HCl was added to adjust pH to 2.0. The solid precipitatedwas collected by filtration, washed with water and dried. The solid wastriturated in hot EtOAc (1 L). After cooling to room temperature andfiltration, the compound of Formula (I) was obtained as a tan solid(18.5 g, 50%). MS [M+H]⁺ found 297.0. ¹H NMR (500 MHz, DMSO-d₆): 13.71(s, 1H), 12.99 (s, 1H), 8.90 (s, 1H), 8.32 (s, 1H), 7.94 (s, 1H), 7.67(s, 1H).

The thermal properties, crystalline nature, apparent purity and moistureuptake of a 6.0 g batch of the free acid of compound (1) are summarizedin Table 1. Saturation data for compound (1) is shown in FIG. 1.

TABLE 1 Apparent Purity Crystallinity Melting Point AdsorptionDesorption (HPLC) (PXRD) (DSC) (40-90% RH) (90-0% RH) 99.8% Weakly 343°C.^(a) +0.59% −0.96% crystalline ^(a)decomposition

Example 2 Potassium Salt of Compound (1)

The potassium salt of1-(5,6-dichloro-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid wasprepared by suspending the free acid (55 g, 1.7 mol) in EtOH (1.5 L) atreflux temperature with K₂CO₃ (12.79 g, 0.85 mol) in 20 mL water addeddropwise over 5 min. Strong mechanic stirring was required to ensureproper agitation. The suspension was stirred at reflux temperature foreight hours and then cooled to room temperature over five hours. Theprecipitated solid was collected by filtration and quickly washed with100 mL of water followed by EtOH. The potassium salt was obtained as awhite solid (38 g, 65%). Subsequently, the mother liquor wasconcentrated and the above process was repeated once to give the secondcrop of the potassium salt (13 g, 22%). MS [M+H]⁺=297.0. ¹H NMR (500MHz, DMSO-d₆): 8.65 (s, 1H), 7.96 (s, 1H), 7.57 (s, 2H).

The potassium salt as prepared by the above re-slurry methodology isnon-hygroscopic and consistent with a poorly crystalline hydrate as seenby PXRD and thermal analysis. Two broad endothermic peaks of thepotassium salt of compound (1) were seen by DSC that can be associatedwith a dehydration event and melt/decomposition, respectively (Table 2).

TABLE 2 Melting Apparent Crystallinity Point Adsorption DesorptionPurity (HPLC) (PXRD) (DSC) (40-90% RH) (90-0% RH) 100.0% crystalline277° C.^(d) +0.53% −0.89% monohydrate ^(d)decomposition

Example 3 Sodium Salt of Compound (1)

The sodium salt of compound (1) is a poorly crystalline, hydrated solidas shown by PXRD and thermal analysis. The DSC reveals two broadendothermic peaks; the first event is associated with a loss of water(˜9% by TGA), while the second endotherm is caused bymelting/decomposition of the salt. The sodium salt was prepared in amethod similar to that used to prepare the potassium salt (slurrymethod).

Example 4 Crystallization Procedure of the Tromethamine Salt of Compound(1)

Two forms of the tromethamine salt have been produced to date. The firstform was obtained from the slurry of compound (1) and tromethamine inaqueous ethanol (14% water). Although not a salt, this physical mixturewas not pursued. The second form was produced from an aqueous workupcontaining excess amounts of counterion. This form was a hydrated saltthat was observed to have a lower apparent aqueous solubility than thepotassium salt. This compound also exhibited poor bulk properties.

Example 5 Crystallization procedure of the meglumine salt of compound(1)

A clear solution (30 mg/mL) of the free acid of compound (1) and 1.2molar equivalent of meglumine was produced in aqueous methanol (12%water) following slight heating. Room temperature stirring with seedingor refrigeration with or without seeding consistently led tocrystallization of the meglumine salt, which was collected viafiltration. This methodology was used to produce a 2 g batch of thesalt. The solvent composition in the above procedure was modified toaqueous ethanol and used by the PDMS API SM Development team to produce8.7-kg of GMP-grade material in support of FIH-enabling and FIH studies.

The thermal properties, crystalline nature, and apparent purity of themeglumine salt of compound (1) are summarized in Table 3. FIGS. 2 and 3show salt bulk properties of the meglumine salt of compound (1),including PXRD data and DSC, TGA and x-ray data, respectively. Thesingle crystal data confirmed the meglumine salt of compound (1) to be adihydrate with the simulated powder pattern in excellent agreement withthe experimental powder pattern as shown in FIGS. 4A and 4B. Themeglumine salt of compound (1) was observed to have improved bulkproperties, solubility, and enhanced processability compared to the freeacid or the potassium salt of compound (1).

TABLE 3 Apparent Purity Crystallinity Melting Sample ID (HPLC) (PXRD)Point (DSC) Meglumine salt of >99.9% crystalline dihydrate 80° C.compound (1)

Example 6 Topical Formulations of Meglumine Salt of Compound (1)

Materials and excipients that used in the development of topicalformulations of a meglumine salt of compound (1) are listed in Table 4.

TABLE 4 Materials Meglumine salt of compound (1) HydroxypropylMethylcellulose (HPMC K15M) Poloxamer 407* Methylcellulose (MC) PEG4000Meglumine (NMDG) Vitamin E-TPGS Carbomer 941 Carbomer 934PCarboxymethylcellulose (Na-CMC) HP-β-CD Sterile Water for Irrigation*solubilization at 5° C. due to the thermo-reversible property of theexcipient.

Table 5 lists the physical and chemical stability results performed onselected formulations of the meglumine salt of compound (1) after 4weeks of storage.

TABLE 5 Meglumine salt of compound (1) Formulation remaining (%)Experiment Composition 2° C. 20° C. 40° C. 1 0.5% HPMC 99.89 101.14101.44 K15M (pH 8.33) 2 1.0% HPMC 99.59 99.21 99.08 K15M (pH 8.33) 32.0% 98.49 99.71 97.20 Methylcellulose (pH 8.32) 4 15% Poloxamer 99.19100.03 87.29* 407 (pH 8.10) 5 20% Poloxamer 97.60 99.57 96.43* 407 (pH8.03) 6 VitE- 100.27 100.32 98.87 TPGS/PEG4000/water (20:20:60; pH 8.06)*Precipitation observed in the vial

HPLC analysis showed that the six formulation compositions werechemically stable for four weeks under the studied storage conditions.The apparent loss of mass balance for the poloxamer-based formulation(Experiment 4) was attributed to the precipitation of the free acid ofcompound (1) at 40° Celsius and not degradation. At 40° Celsius, thepolymers degraded that resulted in the formation of acetic acid,aldehydes, and a concurrent loss of viscosity and drop in pH. Theresulting acidic environment caused precipitation of the insoluble freeacid of compound (1) and discoloration of the formulation. Theinstability of poloxamer (or lutrol) is known and has been disclosed inErlandsson, B., 2002, Polym. Degrad. and Stab., 78:571-575. Such adegradation was not observed in samples stored at room temperature orrefrigerated.

The results of the solubility screen of formulations of the megluminesalt of compound (1) are shown in Table 6. Four of the ten vehiclesinvestigated, namely 1% Na-CMC (Experiment 1), 1% Carbomer 941(Experiment 2), 1% Carbomer 934P (Experiment 3) and 20% HP-β-CD(Experiment 4), did not meet the targeted solubility criterion of 10mg/mL (free acid equivalent) or were toxic to Hela cells. The 1% MCvehicle did not have notable advantages over the product containing 2%MC. The meglumine salt of compound (1) was sufficiently soluble withinan acceptable pH range (6-8.5) in Experiments 5 and 7-11.

TABLE 6 Experiment Formulation Composition Compound (1) Solubility 1  1% Na-CMC  <10 mg/mL 2   1% Carbomer 941  <10 mg/mL 3   1% Carbomer934P  <10 mg/mL 4 0.5% HPMC K15M ≧10 mg/mL 5 1.0% HPMC K15M ≧10 mg/mL 6  1% Methylcellulose ≧10 mg/mL 7   2% Methylcellulose ≧10 mg/mL 8  15%Poloxamer 407 ≧10 mg/mL 9  20% Poloxamer 407 ≧10 mg/mL 10VitE-TPGS/PEG4000/water ≧10 mg/mL (20:20:60) 11  20% HP-β-CD* ≧10 mg/mL*Formulation was toxic to the Hela cells

F) Biological Examples Cellular Assay for HIF1-α

Hela cells (ATCC, Manassas, Va.) were plated in 96-well plates at 20,000cells per well in 100 μl of DMEM containing 10% fetal bovine serum, 1%non-essential amino acids, 50 IU/mL of penicillin and 50 μg/mL ofstreptomycin (all cell culture reagents from Invitrogen, Carlsbad,Calif.). 24 hours after plating, changed media to 100 μl of DMEM without10% fetal bovine serum, 1.1 μl of the stock solution for each compoundwas added and incubated for six hours. All compounds were tested with afinal compound concentration of 100 μM. The supernatant was removed andthe cells were lysed in 55 μl of MSD lysis buffer containing proteaseinhibitors. 50 μl of the cell lysate was then transferred to a blockedMSD human HIF-1α detection plate (Meso-Scale Discovery, Gaithersburg,Md., as per manufacturers protocol), and incubated at room temperatureon an orbital shaker for two hour. After three washes in PBS, 25 μl of20 nM anti-HIF1α detection antibody was added and incubated for 1 hourat room temperature on an orbital shaker. After three washes in PBS, 150μl of 1× read buffer was added and the plate was then read on a MSDSECTOR instrument. Data was analyzed by determining the percent of HIFstimulation in the presence of 100 μM compound relative to an assaycontrol compound,7-[(4-Chloro-phenyl)-(5-methyl-isoxazol-3-ylamino)-methyl]-quinolin-8-ol.This biological data for the meglumine salt of compound (1) is presentedin FIG. 5.

Additional biological data for formulations of the meglumine salt ofcompound (1) is presented in FIGS. 6 through 8.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations and/or modifications as come withinthe scope of the following claims and their equivalents.

1. A formulation comprising the meglumine salt of1-(5,6-dichloro-1H-benzo[d]imidazol-2-yl)-1H-pyrazole-4-carboxylic acid.2. A compound of the formula

in the form of a meglumine salt.
 3. A pharmaceutical compositioncomprising the compound of claim 2 and a pharmaceutically acceptableexcipient.
 4. The formulation of claim 1, wherein said meglumine salt isin the form of a dihydrate.
 5. A topical ointment comprising theformulation of claim
 1. 6. A compound as claimed in claim 2, whereinsaid meglumine salt is a dihydrate.